Global epigenetic and metabolic changes accompany the ...27 Abstract 28 To further understand the...

Global epigenetic and metabolic changes accompany the

alterations in fruit size and shape of Cucurbita pepo L. intra-

species grafting

Aliki Xanthopoulou 1 , Aphrodite Tsaballa 2 , Ioannis Ganopoulos 3 , Aliki Kapazoglou 1 , Evangellia Avramidou 4

, Filippos Aravanopoulos 4 , Theodoros Moysiadis 1 , Maslin Osathanunkul 5 , Athanasios Tsaftaris 6 , Andreas

Doulis 7 , Apostolos Kalivas 3 , Eirini Sarrou 3 , Stefan Martens 8 , Irini Nianiou- Obeidat 9 , Panagiotis MadesisCorresp. 10

1 Institute of Applied Biosciences, Institute of Applied Biosciences/CERTH, Thermi Thessaloniki, Greece

2 School of Biological Sciences, University of East Anglia, Norwich, UK., School of Biological Sciences, University of East Anglia, Norwich, UK., Norwich,

United Kingdom3 Hellenic Agricultural Organization DEMETER (ex NAGREF), Institute of Plant Breeding and Genetic Resources, Thermi, Thessaloniki, Greece

4 Faculty of Forestry and Natural Environment, Laboratory of Forest Genetics and Tree Breeding, Aristotle University of Thessaloniki, Thessaloniki, Greece

5 Chiang Mai University, Department of Biology, Faculty of Science,, Chiang Mai, Thailand

6 American Farm School, Perrotis College, Thessaloniki, Greece

7 Hellenic Agricultural Organization DEMETER (ex NAGREF),, Institute of Olive Tree, Subtropical Crops and Viticulture, Heraklion, Greece

8 IASMA Research and Innovation Centre, Fondazione Edmund Mach (FEM),, Department of Food Quality and Nutrition Department, San Michele all' Adige,

Italy9 Department of Genetics and Plant Breeding, Aristotle University of Thessaloniki, Thessaloniki, Greece

10 Institute of Applied Biosciences, Institute of Applied Biosciences/CERTH

Corresponding Author: Panagiotis Madesis

Email address: [email protected]

To further understand the impact of grafting on fruit characteristics and to comprehend

the mechanisms involved in graft-induced changes we studied homo- and hetero- grafted

Cucurbita pepo cultivars (cv.) that vary in fruit size and shape. C. pepo cv. ‘Munchkin’

(small fruit) and cv. ‘Big Moose’ (large fruit) as well as cv. ‘Round green’ (round fruit) and

cv. ‘Princess’ (elongated fruit) were homografted and reciprocally heterografted. The

results show significant changes in fruit size when ‘Big Moose’ was grafted onto ‘Munchkin’

rootstocks in comparison to homo-grafted controls. Moderate changes in fruit shape were

observed when grafting of cv. ‘Round green’ and cv. ‘Princess’ were performed. This is the

first report of such phenotypic changes after intra-species/inter-cultivar grafting in

Cucurbitaceae. Additionally, we found significant changes in i) secondary metabolite

profile, ii) global DNA methylation pattern and iii) miRNA expression patterns in grafted

scions and iv) DNA methylation on graft-induced phenotypic changes in grafted plants. Our

results contribute to further understanding graft-induced effects on fruit morphology in

intra-species grafting. Furthermore, our results pave the way for understanding the role of

phenolic metabolites and epigenetic molecular mechanisms on the phenotypic changes

recorded.

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.3389v1 | CC BY 4.0 Open Access | rec: 3 Nov 2017, publ: 3 Nov 2017

1 Global epigenetic and metabolic changes accompany the alterations in fruit size and shape

2 of Cucurbita pepo L. intra-species grafting

3 Aliki Xanthopoulou1,2, Aphrodite Tsaballa3, Ioannis Ganopoulos4,, Aliki Kapazoglou2, Evangelia

4 Avramidou5, Filippos A. Aravanopoulos5, Theodoros Moysiadis2, Maslin Osathanunkul9,

5 Athanasios Tsaftaris1,2,8 Andreas G. Doulis6, Apostolos Kalivas4, Eirini Sarrou4, Stefan Martens7,

6 Irini Nianiou- Obeidat1*, Panagiotis Madesis2*

71Aristotle University of Thessaloniki, Department of Genetics and Plant Breeding, Thessaloniki,

8 GR-54124, Greece

92Institute of Applied Biosciences (INAB), CERTH, Thermi-Thessaloniki, GR-57001, Greece

103School of Biological Sciences, University of East Anglia, Norwich, UK.

114Institute of Plant Breeding and Genetic Resources, Hellenic Agricultural Organization

12 DEMETER (ex NAGREF), Thermi, Macedonia, GR-57001, Greece

135Aristotle University of Thessaloniki, Faculty of Forestry and Natural Environment, Laboratory

14 of Forest Genetics and Tree Breeding, GR-54124, Greece

156Hellenic Agricultural Organization DEMETER (ex NAGREF), Institute of Olive Tree,

16 Subtropical Crops and Viticulture, Heraklion, Crete, GR-71003, Greece.

177Department of Food Quality and Nutrition Department, IASMA Research and Innovation

18 Centre, Fondazione Edmund Mach (FEM), Via E. Mach 1, 38010 San Michele all' Adige, (TN),

19 Italy

208Perrotis College, American Farm School. Thessaloniki, GR-57001. Greece

219Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200,

22 Thailand

23

24 Corresponding authors: [email protected] and [email protected]

25


https://www.researchgate.net/institution/University_of_East_Anglia/department/School_of_Biological_Sciences

https://www.researchgate.net/institution/University_of_East_Anglia

mailto:[email protected]

mailto:[email protected]

27 Abstract

28 To further understand the impact of grafting on fruit characteristics and to comprehend the

29 mechanisms involved in graft-induced changes we studied homo- and hetero- grafted Cucurbita

30 pepo cultivars (cv.) that vary in fruit size and shape. C. pepo cv. ‘Munchkin’ (small fruit) and cv.

31 ‘Big Moose’ (large fruit) as well as cv. ‘Round green’ (round fruit) and cv. ‘Princess’ (elongated

32 fruit) were homografted and reciprocally heterografted. The results show significant changes in

33 fruit size when ‘Big Moose’ was grafted onto ‘Munchkin’ rootstocks in comparison to homo-

34 grafted controls. Moderate changes in fruit shape were observed when grafting of cv. ‘Round

35 green’ and cv. ‘Princess’ were performed. This is the first report of such phenotypic changes

36 after intra-species/inter-cultivar grafting in Cucurbitaceae. Additionally, we found significant

37 changes in i) secondary metabolite profile, ii) global DNA methylation pattern and iii) miRNA

38 expression patterns in grafted scions and iv) DNA methylation on graft-induced phenotypic

39 changes in grafted plants. Our results contribute to further understanding graft-induced effects on

40 fruit morphology in intra-species grafting. Furthermore, our results pave the way for

41 understanding the role of phenolic metabolites and epigenetic molecular mechanisms on the

42 phenotypic changes recorded.

43

44 Keywords: Cucurbitaceae, MSAP methylation sensitive amplified polymorphisms markers,

45 epigenetic, phenolics, miRNAs, fruit morphology

46

47

48

49


50 Introduction

51 Grafting is an important cultivation technique in agriculture, employed widely to improve

52 performance of important horticultural crops such as woody fruit trees, grapes and lately on

53 vegetable crops (Goldschmidt 2014; Mudge et al. 2009). It is an ancient technique used for over

54 2,000 years that was first applied in woody species for improving yield and disease resistance

55 (Kubota et al. 2008). Vegetable grafting which originated in the early 20th century, involves

56 mainly Cucurbitaceae and Solanaceae species and has been utilized extensively for enhancing

57 fruit quality, resistance to diseases and tolerance to abiotic stresses (Goldschmidt 2014; Mudge et

58 al. 2009). Grafting was originally employed to avoid the detrimental effects of soil borne

59 pathogens infecting the roots of susceptible plants through the use of resistant rootstocks grafted

60 onto the plant of interest (King et al. 2008; Louws et al. 2010). Selected beneficial rootstocks can

61 provide resistance to bacteria, nematodes, fungi, as well as enhanced tolerance to abiotic stresses

62 such as extreme temperatures, increased salinity, increased calcium and heavy metals in the soil

63 (Colla et al. 2010; King et al. 2008).

64 Whereas horticulturists were trying to improve crops through grafting in order to provide

65 higher biomass performance and resistance in plants’ diseases and biotic stress, the quality

66 (nutritional/organoleptic properties) of product didn’t attract much attention. Nowadays such

67 kind of improvement strategies seem to focus also on some specific compounds contributing to

68 the nutritional value of vegetables, such as polyphenols, carotenoids, volatiles etc. Polyphenols,

69 as products of secondary metabolism, have received much attention the last decades, due to their

70 antioxidant (Valenzuela et al. 2014), antimicrobial (Nascimento et al. 2014), antiviral (Katayama

71 et al. 2013) bioactivities among others. In the past decade, a great number of studies regarding

72 Cucurbitaceae grafting aimed at improving fruit quality and plant vigour of economically

73 important vegetables such as cucumber, melon and watermelon (Cohen et al. 2007). Grafting of

74 watermelon onto selected rootstock genotypes resulted in significant increase of lycopene

75 content in watermelon fruits in addition to enhanced resistance to soil-borne diseases (Mohamed

76 et al. 2012), whereas different rootstock-scion combinations led to alterations in the content of

77 plant nutrients in leaves and fruits of watermelon grafts (Yetisir et al. 2013). Moreover,

78 cucumber and melon grafted onto specific pumpkin rootstocks led to altered organoleptic

79 properties in addition to enhanced tolerance to high salinity (Huang et al. 2013; Orsini et al.

80 2013; Rouphael et al. 2012). Numerous complex processes occur during grafting, including


81 formation of the graft union and the common vasculature, transport of information from

82 rootstock to scion and vice versa, as well as the differing effects of scion and rootstock genotype

83 combinations. Thus, it is reasonable to assume multiple molecular mechanisms operating

84 throughout the growth of a grafted plant. These mechanisms include transport of important

85 information molecules such as small RNAs as well as epigenetic effects, such as methylation of

86 DNA, both of which can, in turn, affect gene expression. Trafficking of macromolecules such as

87 proteins and of genetic material or hormones and nutrients, have been demonstrated in many

88 instances between grafting partners (Albacete et al. 2015). Regarding movement of genetic

89 material, an increasing number of reports implicate long distance transport of RNA molecules

90 such as messenger and small RNAs. Movement of RNA molecules over long distances, across

91 plant organs and through the phloem has been well documented (Kalantidis et al. 2008; Kehr &

92 Buhtz 2008; Lough & Lucas 2006; Lucas et al. 2001; Mermigka et al. 2015; Omid et al. 2007;

93 Spiegelman et al. 2013). Similarly, transport of small non-coding RNAs (siRNAs and miRNAs)

94 across graft segments of homo-grafted and hetero-grafted plants has been demonstrated in a

95 number of studies in Arabidopsis, Solanaceae and Cucurbitaceae (Haroldsen et al. 2012; Melnyk

96 et al. 2011; Palauqui et al. 1997).. In many occasions mobile RNA molecules have a

97 transcriptional or post-transcriptional gene silencing (Haroldsen et al. 2012; Melnyk et al. 2011;

98 Palauqui et al. 1997). It is known that miRNAs play crucial roles in all plant biological

99 processes, including growth, vegetative and reproductive development, and response to abiotic

100 and biotic stresses (Barrera-Figueroa et al. 2013; Li et al. 2016; Zhang 2015). MiRNAs are post-

101 transcriptional gene regulators that target mRNAs and either cleave the transcript or inhibit

102 translation (Dong et al. 2013). They target a wide range of transcription factors related to

103 developmental processes as well as genes that code for enzymes involved in metabolic pathways.

104 Several evolutionary conserved families of miRNAs such as the miR156, miR159, miR165/166,

105 mirR171, miR172, control various aspects of development in plants. MiR156 has been

106 associated with changes in leaf biomass as its overexpression led to significant increase in total

107 leaf number and subsequent plant biomass in Arabidopsis (Schwab et al. 2005).

108 Global DNA methylation studies, using either methylation sensitive amplified

109 polymorphisms (MSAP) or locus-specific bisulfite sequencing analyses revealed significant

110 alterations in the DNA methylation pattern of grafted plants after reciprocal inter-species grafting

111 among three Solanaceae species (Wu et al. 2013). In particular, the global DNA methylation


112 pattern of tomato, eggplant and pepper scions was shown to be extensively altered in two DNA

113 methylation contexts (CG and CHG) upon grafting. Importantly, the graft-induced methylation

114 changes were inherited in the self-pollinated progeny, indicating that grafting can induce

115 epigenetic changes that can be stably transferred to subsequent generations (Wu et al. 2013). In

116 agreement with the study above, our group has recently demonstrated altered DNA methylation

117 patterns upon inter-species hetero-grafting of three Cucurbitaceae species 33. In particular, DNA

118 methylation levels were significantly increased in melon and cucumber scions grafted onto

119 pumpkin rootstocks (Avramidou et al. 2014).

120 Fruit morphology, which includes fruit shape and size, is another important trait of

121 agronomic importance in vegetable species of the Solanaceae and Cucurbitaceae families.

122 However, although studies during the past decades have focused on fruit yield and quality

123 (texture, aroma, flavor, nutrients), resistance to diseases and tolerance to abiotic stresses, little is

124 known about the effect of grafting on fruit morphology and the underlying molecular

125 mechanisms governing this process. A few reports have described an effect on fruit shape upon

126 intra-species grafting in pepper (Capsicum annuum) (Taller et al. 1999; Tsaballa et al. 2012;

127 Yagishita & Hirata 1987). Tsaballa et al. (2012) 35 have demonstrated remarkable effects on fruit

128 shape of the scion partner in a rootstock–scion combination of two different pepper genotypes

129 with different fruit shape. Specifically, grafting of the round-fruit shaped cv. ‘Round’, onto a

130 long-fruit shaped cv. ‘Long’, resulted in substantially elongated fruits in the scion cv. ‘Round’

131 plants (Tsaballa et al. 2012). However, there are no reports, to date, concerning the effects of

132 grafting on fruit morphology in Cucurbitaceae. In order to further investigate grafting in

133 Cucurbitaceae and the effect of rootstock-scion combinations in fruit size and shape we have

134 applied reciprocal hetero-graftings of two different C. pepo cvs., ‘Big Moose’ and ‘Munchkin’,

135 with different fruit sizes as well as two other C. pepo cvs., ‘Princess’ and ‘Round green’, with

136 different fruit shapes. In addition, we have assessed changes in DNA methylation patterns

137 employing comparative MSAP methodology which allows for the documentation of epigenetic

138 alterations between different crosses and between plants bearing fruits of different sizes and

139 morphology. Further, we have analyzed the expression of several miRNAs in order to probe

140 variations of these epigenetic modifiers linked to the grafting process and possibly with grafting-

141 induced fruit morphological changes. Additionally, we determined concentrations of a series of

142 selected secondary metabolites and compared thus produced metabolic profiles between different


143 grafting combinations and non-grafted controls. We report here graft-induced changes in fruit

144 size and moderate changes in fruit shape of C. pepo scions accompanied by significant changes

145 in scion genomic DNA methylation, patterns, abundance of specific miRNA molecules and

146 secondary metabolites as compared to non-grafted control tissues.

147

148 Materials and Methods

149 Plant Material

150 Two independent grafting and MSAP experiments were conducted during two

151 consecutive summer periods (2014 and 2015). Samples from 2014 were used for MSAP analysis

152 in relation to fruit size, whereas samples from 2015 were used for MSAP analysis in relation to

153 fruit shape and metabolite profile.

154 Four Cucurbita pepo cultivars were used for grafting; (1) cv. ‘Round green’ (round fruit

155 shape) (2) cv. ‘Princess’ (long fruit shape) (3) cv. ‘Big Moose’ (large fruit size) and (4) cv.

156 ‘Munchkin’ (small fruit size). Seeds were initially sown in small pots that were kept in the

157 greenhouse at 25 °C. Grafting was carried out by the time plants had 2–3 true leaves and a shoot

158 diameter of about 1.5–2.0 mm. In total, four grafting combinations were produced representing

159 two reciprocal cv. combinations as following: (1) cv. ‘Round green’ on cv. ‘Princess’, (2) cv.

160 ‘Princess’ on cv. ‘Round green’, (3) cv. ‘Big Moose’ on cv. ‘Munchkin’ and (4) cv. ‘Munchkin’

161 on cv. ‘Big Moose’. Grafting was performed following the splice-grafting method employing

162 grafting clips (for a review of grafting methods see (Lee et al. 2010)). A total of 4 plants from

163 each grafting combination i.e. rootstock (RS) x scion (SC) were used for scoring for either fruit

164 size or fruit shape as well as for MSAP analysis. Controls included were (1) the homo-grafted

165 plants for example cv. ‘Round green’ grafted onto cv. ‘Round green’ for all four cvs. and (2) the

166 non-grafted s that served as reference state in MSAP comparisons as well (see below). Following

167 grafting, all plants were transferred in the growth chamber under constant temperature of 25 °C,

168 RH 85–95%, light flux 2,000 lux, photoperiod of 16 h light/8 h dark and kept for 2 weeks.

169 Successfully grafted plants were transferred in the greenhouse for an acclimation period of one

170 month, transferred to 7 L pots filled with calcareous clay soil afterwards and allowed to grow in

171 the open while watered regularly with nutrient solution. The same grafting design was used both

172 years.

173


174 DNA extraction and ISSR molecular analysis

175 DNA extraction from leaves of controls and scions, from plants presenting statistically

176 different phenotypes were used, was performed using NucleoSpin Plant II Kit (Macherey Nagel,

177 Duren, Germany), according to manufacturer's instructions.

178 For the detection of putative intra-cultivar variation, we selected 6 genotypes of each

179 grafting cultivar combinations (72 individuals in total) and we used a set of six Inter Simple

180 Sequence Repeats (ISSR) primers as described previously by Xanthopoulou et al. (2015). PCR

181 reactions and gel electrophoresis were composed according to the authors above.

182

183 Methylation Sensitive Amplification Polymorphism (MSAP) analysis

184 For each sample, MSAP analyses were performed using both EcoRI/HpaII and

185 EcoRI/MspI digests. Digestion of 200-ng aliquots of genomic DNA with either of the two

186 isoschizomers (HpaII, MspI) was carried out in 20 μl containing 1Χ one for all Buffer, 4 U

187 EcoRI (New England Biolabs, Ipswich, MA, USA) and 3 U of either HpaII or MspI enzyme

188 (New England Biolabs, Ipswich, MA, USA) for 3 h at 37°C. Two different adapters, designed to

189 avoid reconstruction of restriction sites, one for the EcoRI sticky end and one for the HpaII/MspI

190 sticky end, were ligated to the DNA after digestion, by adding to each final digestion 5 μl of a

191 mix containing 5 pmol of EcoRI adapter, 50 pmol of HpaII/MspI adapter, 1 mM ATP, 1Χ one

192 for all Buffer, and 1 U of T4 DNA ligase (Invitrogen, CA, USA). The ligation was incubated for

193 3 h at 25°C. The EcoRI adapter consisted of the combination of two primers: 5Ά-

194 CTCGTAGACTGCGTACC-3Ά and 3Ά-CTGACGCATGGTTAA-5Ά. EcoRI and HpaII/MspI

195 adapters are presented in Table S1. Digested and ligated DNA fragments were diluted 5-fold and

196 used as templates for the pre-amplification reaction. Two different pre-amplification reactions

197 were performed using EcoRI/MspI-A and EcoRI/HpaII-A primers in a total volume of 20 μl

198 containing 1X Kapa Taq Buffer, 0.2mM of each dNTP, 2.5 mM MgCl2, 30 ng of each primer

199 EcoRI+A, HpaII/MspI+A, 1U Taq DNA polymerase (Kapa Biosystems, Woburn, MA) and 5 μl

200 of diluted fragments. PCR amplifications were carried out in a BioRad thermocycler for 23

201 cycles of 30 s at 94°C, 30 sec at 56°C, and 1 min at 72°C. Pre-amplified fragments were diluted

202 10-fold and used as templates for the selective amplification. Οne labelled EcoRI and one

203 HpaII/MspI primer, with the same sequences as those used for the pre-amplification but with two

204 and three selective nucleotides respectively at the 3 end, were used in each analysis. PCR was


205 performed in a total volume of 10 μl including 1X Kapa Taq Buffer 2.5 mM MgCl2, 0.08 mM of

206 each dNTP, 5 ng of labeledEcoRI primer, 30 ng of HpaII/MspI primer, 1 U of Taq DNA

207 polymerase (Kapa Biosystems), and 3 μl of diluted pre-amplified DNA. The selective

208 amplification was carried out using classical AFLP cycling parameters (Vos et al. 1995),

209 employing eight different primer combinations (Table S1).

210

211 MSAP data collection and processing

212 MSAP product mixtures were denatured in formamide at 94°C for 2 min and

213 electrophoretically fractionated on an ABI Prism 3730xl (Applied Biosystems, Carlsbad, CA,

214 USA). In order to reduce the impact of potential size homoplasy only fragments ranging from

215 150 to 500 bases in size were counted (Vekemans et al. 2002). Fragment size was determined

216 using Genemapper v4.0 (Applied Biosystems, Carlsbad, CA, USA) employing an internal

217 standard (GS 500 LIZ, Applied Biosystems) and an initial data matrix was produced with allele

218 presence scored as “1” and absence as “0”.

219 Our scoring initially entailed determining marker presence / absence between a seedling

220 (reference state) and grafted plants of the same cv. even if a single band in one of the two

221 isoschizomer profiles of a single individual was detected. Scions from “homo-” or “hetero-”

222 grafted plants were scored independently and were considered as two different treatment states.

223 The same individual control seedlings were considered as reference states in comparisons

224 involving scions of the same cv. irrespective of rootstock cv. (i.e. irrespective of “homo-” or

225 “hetero-” treatment states). Each methylation state was described with two binary digits (duplets)

226 corresponding to the two isoschizomer profiles. Quadruplets (a series of four binary digits) were

227 subsequently, produced by pooling together duplets from the reference and from the treatment

228 state (two different treatment states in total; “homo-” and “hetero-”) and were assigned to state

229 change events between reference and treatment states. Sixteen alternate quadruplets could thus

230 be produced each grouped according to (Haoa et al. 2004) as de novo methylation (three events),

231 demethylation (three events), other (ambiguous or too complex pattern to be explained; four

232 events) or no change (six events; not specifically enumerated) (Haoa et al. 2004). All data

233 management steps, from initial matrix formation till event change classification and tallying

234 were performed employing an in-house script. Only polymorphic fragments (more than 5%

235 present or absent) were retained for subsequent analysis (MSAP polymorphic loci). Once the


236 quadruplets of each sample (grafting combination) have been derived (by comparison to

237 respective non-grafted controls) they were categorized according to deduced methylation change

238 event (de-methylation, de novo methylation, no change, other-non classifiable). The number of

239 each one of the four change events -per sample- was converted to percentages against the

240 number of total events of each sample separately. Four different percentages are produced per

241 each sample corresponding to the four different change events. Comparisons and statistical

242 analyses of such percentage across samples could thus be made (Figures 3 and 4). Change event

243 “other” is not portrayed in Figures 3 and 4). Subsequently, statistically significant differences

244 between the two treatment states i.e. “homo-“ vs. “hetero-“ grafted plants were assessed for the

245 MSAP polymorphic loci by means of a t-test employing SPSS (ver. 22). Separate tests were

246 performed for each cv. serving as rootstock (four independent experiments / statistical analyses

247 in total) and the two combinations employing the self cv. as scion.

248

249 miRNA expression profile via stem-loop RT-PCR

250 Total RNA was extracted from leaves (from plants presenting statistically different

251 phenotypes) using the TRIzol® Reagent (Invitrogen, USA) and DNase I was then used to

252 remove DNA. The integrity and purity of the total RNA were evaluated using 2% denaturing gel

253 electrophoresis and NanoDrop DU8000 spectrophotometry (A260/A280 and A260/A230). The

254 expression profiles of five miRNAs were assayed by stem–loop reverse transcription-PCR (RT–

255 PCR). A 200 ng aliquot of total RNA was used for the initiation of the reverse transcription

256 reaction. The stem–loop reverse transcription primers were designed following the method

257 described by (Varkonyi-Gasic et al. 2007). The reverse transcription product is amplified using a

258 miRNA-specific forward primer and a universal reverse primer. The stem–loop reverse

259 transcription reactions were performed by Superscript III reverse transcriptase (Life

260 Technologies, Carlsbad, CA, USA) transcriptase according to the supplier's manual. PCR

261 primers were then added to perform the PCR. Relative quantification was performed using

262 Elongation Factor-1 (EF-1) as the endogenous control reference gene (Obrero et al. 2011). The

263 data were analyzed using the REST software (Pfaffl et al. 2002).

264

265 Phenolic metabolite analysis

266 Fresh collected samples (each sample obtained from pooling fruit material from three


267 individual plants) were freeze dried (Freeze-dryer Alpha 1-2 LD plus, Christ, Germany; at -

268 24°C), and then pulverized to fine powder. The extraction was performed by mixing 100 mg of

269 freeze-dried fruit sample with 5 mL 80% methanol into 15 mL falcon tube. The samples and

270 solvent were mixed by orbital shaker for 3 h at room temperature and the extraction proceeded

271 overnight at 4 °C in the dark. The resulting solutions were filtered on a 0.22 µm PFTE membrane

272 into a glass vial and analyzed as described below. Three replicates for each sample were done.

273 The analysis of phenolic compounds was performed using the method described previously

274 by (Vrhovsek et al. 2012). Samples were directly injected after extraction. Targeted Ultra

275 Performance Liquid Chromatography was performed on a Waters Acquity UPLC system

276 (Milford, MA, USA) consisting of a binary pump, an online vacuum degasser, an autosampler,

277 and a column compartment. Separation of the phenolic compounds was achieved on a Waters

278 Acquity HSS T3 column 1.8 μm, 100 mm × 2.1 mm (Milford), kept at 40 °C.

279 Mass spectrometry detection was performed on a Waters Xevo TQMS (Milford)

280 instrument equipped with an electrospray (ESI) source. Data processing was performed using the

281 Mass Lynx Target Lynx Application Manager (Waters).

282

283 Results

284

285 Graft-induced changes in fruit size and shape

286 Two C. pepo cultivars differing in fruit size, cv. ‘Munchkin’, a small fruited cultivar, and

287 cv. ‘Big Moose’, a large fruited cultivar (Figure 1A) and two cultivars differing in fruit shape,

288 cv. ‘Round green’, a round fruited cultivar and cv. ‘Princess’, an elongated fruited cultivar

289 (Figure 2A), were used to assess possible graft-induced alterations in fruit morphology in

290 reciprocal hetero-grafting combinations. Fruit morphology of grafted plants was assessed using

291 morphological traits according to UPOV descriptors such as weight, polar diameter and

292 equatorial diameter for the scion fruits from each grafting combination as well as from control

293 (non-grafted). The majority of the fruits produced by the ‘Munchkin’ and ‘Big Moose’ grafting

294 combinations although they had the expected phenotype i.e. they were similar to the fruit

295 phenotype of the non-grafted scion in every case (Figure 1B), they presented statistically

296 significant phenotypic differences with the fruits from the non-grafted or homografted plants in

297 terms of weigh, equatorial and polar diameter. In addition a single grafted ‘Munchkin’ (RS) x


298 ‘Big Moose’ (SC) plant (termed “with altered fruit size”; Table 1) produced fruits with

299 pronounced morphological differences when compared to the non-grafted ‘Big Moose’ plants

300 (Figure 1C). A Distinct reduction in fruit weight and fruit size in this plant could be observed

301 when compared to the rest of the fruits produced by this grafting combination (Figure 1D).

302 Further, the reduction in fruit weight was approximately 2.6-fold compared to ‘Big Moose’ non-

303 grafted control, more than 3-fold compared to ‘Big Moose’ homografts and 2.4-fold compared to

304 the majority of the ‘Munchkin’ (RS) x ‘Big Moose’ (SC). In addition, a significant decrease in

305 both equatorial and polar fruit diameter of about 2-fold was evidenced in this heterograft as

306 compared to the control non-grafted ‘Big Moose’ and Munchkin’ (RS) x ‘Big Moose’ (SC)

307 plants (Table 1).

308

309 On the other hand the reciprocal combination, ‘Big Moose’ (RS) x ‘Munchkin’ (SC) did

310 not produce any discrete evident morphological changes (i.e. in fruit size) like the one before

311 (Figure 1B-bottom) (Table 1). However, moderate changes statistically significant were observed

312 in fruit shape in the reciprocal combinations of ‘Round green’ and ‘Princess’. ‘Princess’ (RS) x

313 ‘Round green’ (SC) resulted in less round fruits and the reverse combination, ‘Round green’

314 (RS) x ‘Princess’ (SC) led to fruits that were smaller as compared to the control seedlings

315 (Figure 2B). Grafting combinations of ‘Princess’ and ‘Round green’ did not produce extreme

316 differences in terms of morphological traits yet there were statistically significant especially for

317 equatorial and polar diameter when compared to non-grafted or homografted plants (Table 1).

318 To exclude the possibility that fruit morphology changes rise from genetic variation

319 within cultivars or cross-pollination between them since C. pepo is an openly pollinated species,

320 molecular analysis with six polymorphic Inter simple sequence repeat (ISSR) markers was

321 performed according to (Xanthopoulou et al. 2015). No intra-cultivar variation was found

322 among the 72 examined plants suggesting that epigenetic (rather than genetic) changes

323 associated with grafting should be sought as the underlying mechanism inducing morphological

324 alterations.

325

326 Graft-induced changes in scion global DNA methylation pattern

327 The effect of grafting on the pattern of scion DNA methylation pattern was assessed

328 utilizing a comparative MSAP analysis (Supplementary Figure 1). Initially, the MSAP method


329 can detect the DNA methylation status of 5’-CCGG-3’sites, employing two pairs of

330 isoschizomers EcoRI/HpaIIand EcoRI/MspI that have different sensitivity to methylation at the

331 inner or outer cytosines. For each sample the DNA methylation pattern at 5΄-CCGG-3΄ sites is

332 determined by scoring the presence or absence of marker bands of EcoRI/HpaII and EcoRI /MspI

333 digestion of genomic DNA followed by PCR amplification (Supplementary Figure 1).

334 Differences in PCR products obtained from two different samples reflect different methylation

335 states at the cytosines restriction site. Per sample, methylation patterns are deduced by

336 determining amplification differences between the two pairs of isoschizomers while methylation

337 pattern changes are determined following comparisons between treatments (different grafting

338 combinations) and controls (non-grafted seedlings). We consider the case with four different

339 methylation change events (1. de novo methylation, 2. demethylation, 3. no change, 4. other

340 variation).

341 As a first step, methylation change (MSAP) data were averaged and plot by event and by

342 grafting combination (Figures 3 and 4). In the ‘Munchkin’ (RS) x ‘Big Moose’(SC) combination,

343 which resulted in the significant fruit size reduction, an increase of approximately 5-fold in DNA

344 de novo methylation was observed in the scion leaves compared to the control ‘Big Moose’ non-

345 grafted seedlings (Figure 3A). In addition, an increase of about 2-fold in demethylation was

346 observed in the scions in comparison to the control ‘Big Moose’ non-grafted, whereas 40% of

347 cytosine sites remained unchanged (Figure 3A). In the ‘Big Moose’ (RS) x ‘Munchkin’ (SC)

348 combination an increase in DNA methylation of approximately 2-fold was evidenced in the

349 leaves of the scion plants as compared to the ‘Munchkin’ control seedlings, whereas a change of

350 approximately 8-fold in DNA demethylation levels was detected (Figure 3B). In the ‘Princess’

351 (RS) x ‘Round green’ (SC) graft which resulted to a flat-shaped-fruit, a remarkable increase in de

352 novo DNA methylation of 20-fold is evidenced in the scions as compared to the control ‘Round

353 green’ non-grafted (Figure 4A). A marked increase in DNA demethylation of 12-fold was also

354 observed in the ‘Round green’ scions, as compared to the control ‘Round green’ (Figure 4A).

355 Likewise, in the ‘Round green’ (RS) x ‘Princess’ (SC) graft, which resulted in a smaller fruit

356 compared to the non-grafted seedling, a significant increase in DNA methylation of

357 approximately 16-fold and an increase in DNA demethylation of about 12- fold was observed in

358 ‘Princess’ scions, relative to the control ‘Princess’ non-grafted plants (Figure 4B).

359 As a second step, methylation change data underwent a detailed statistical analysis. The


360 chi-square test was applied to test the null hypothesis that homografting vs heterografting (four

361 independent comparisons-please see a)-d) below) are homogeneous regarding the methylation

362 event, i.e. the respective theoretical distributions of percentages regarding the categories of the

363 methylation event do not differ statistically significantly. The significance level was set to 0.05.

364 We considered the case with four different methylation event categories (1. de novo methylation,

365 2. demethylation, 3. no change, 4. other variation). A significant result would indicate significant

366 overall difference in the methylation categories’ percentages between homografting and

367 heterografting, and therefore statistically significant difference in how methylation is expressed

368 in these two cases. We performed independently four comparisons, i.e. a) ‘Munchkin’ homo

369 versus ‘Big Moose’ x ‘Munchkin’, b) ‘Big Moose’ homo versus ‘Munchkin’ x ‘Big Moose’, c)

370 ‘Princess’ homo versus ‘Round green’ x ‘Princess’ and d) ‘Round green’ homo versus ‘Princess’

371 x ‘Round green’. The results were a) 46.256, p-value<0.001, b) 49.640, p-value<0.001, c)

372 16.215, p-value=0.001 and d) 29.867, p-value<0.001. Therefore, the null hypothesis of

373 homogeneous methylation behaviour was rejected for all comparisons, i.e. all differences

374 considered were statistically significant. In addition to the above comparisons, for each one of

375 the four cultivars, the goodness of fit test was applied to test the null hypothesis that the

376 theoretical percentages regarding the categories of the methylation event are equal, and by that to

377 actually test if the type of methylation event appears at random within each cultivar. The results

378 were i) 307.668, p-value<0.001, ii) 304.480, p-value<0.001, iii) 248.066, p-value=0.001 and iv)

379 120.224, p-value<0.001. Therefore, the null hypothesis of equal methylation categories’

380 percentages was rejected for all comparisons, i.e. the type of change did not appear at random.

381 The Cochran’s criterion for the validity of all the above chi-square based tests has been checked

382 and was satisfied in all cases.

383 Graft induced changes in miRNA expression

384 As depicted in Figure 5A, significant changes were observed in expression levels of a

385 number of miRNAs in the ‘Munchkin’ and ‘Big Moose’ grafted plants, when compared to the

386 non-grafted controls. In the ‘Munchkin’ (RS) x ‘Big Moose’ (SC) that was the combination in

387 the grafted plant with the reduced fruit size, the expression of miR159 and miR164 was

388 significantly decreased by approximately 4-fold, whereas miR171 was decreased by 2-fold and

389 miR166 by 1.6-fold. On the contrary, non-significant expression changes were recorded in the

390 scions of the ‘Big Moose’ (RS) x ‘Munchkin’ (SC) heterograft, except for miR171, which was


391 induced by about 5-fold and miR164, which was 1.5 times more abundant in comparison to the

392 control (Figure 5A). A significant induction of about 2-fold, for miR171 was also observed in the

393 ‘Round green’ (RS) x ‘Princess’ (SC) heterograft, which had a mild effect in fruit shape (Figure

394 5B). A reduction of 1.6-fold was evidenced for miR159 in the ‘Round green’ (RS) x ‘Princess’

395 (SC) heterograft (Figure 5B).

396

397 Phenolic metabolite analysis

398 Fruit samples from all grafting combinations were subjected to targeted LC-MS/MS

399 analysis in order to identify the effect and possible changes on the secondary metabolite level in

400 different grafting combinations. In total, seventeen compounds were identified in C. pepo fruit

401 samples from the size differing cultivars and their grafting combinations while eighteen

402 compounds were identified in the shape differing cultivars and their grafting combinations.

403 Compounds detected varied in presence and concentration, as shown in the heatmaps produced

404 (Figures 6A and B). In the majority of samples, kaempferol-3-O-rutinoside (Km3rut), rutin and

405 isorhamnetin-3-O-rutinoside (Iso3rut) were the most abundant compounds, whereas in the

406 cultivars differing in fruit size significant amounts were detected for p-hydroxybenzoic (p-HBA),

407 cinnamic, ferulic and vanilic acid in scion fruits. In the different cultivars, nine (‘Big Moose’,

408 ‘Princess’), ten (‘Munchkin’) and fourteen (‘Round green’) phenolic metabolites were identified

409 (Tables 2 and 3), while several metabolites seem to be influenced by grafting combination.

410 Concerning the fruit cultivars differing in shape, some of the identified metabolites such as

411 Km3rut, quercetin-3-O-glucoside, rutin and Iso3rut, which are present in the control non grafted

412 and the ‘Big Moose’ homograft as long as in the heterograft ‘Munchkin’ (RS) x ‘Big Moose’

413 (SC), were absent in all the rest of grafting samples. On the other hand, the compounds p-HBA,

414 sinapoyl alcohol and arbutin were present only in the fruits obtained from the ‘Munchkin’ non-

415 grafted plants, its homografts, and the heterografts where ‘Munchkin’ cultivar was used as the

416 scion. Furthermore, it became apparent in the case of the heterograft ‘Princess’ (RS) x ‘Round’

417 (SC) that there was a stimulation in most of the identified metabolites in the cultivars with green

418 fruit colour, especially in the category of rutinosides. Both homografts of ‘Princess’ and ‘Round

419 green’ exhibited a limited level of metabolites.

420

421 Discussion


422 In the current study we investigated the effect of C. pepo intra-species grafting on scion’s

423 fruit size and shape and its association with global epigenetic and metabolic profiles. The

424 heterograft of two C. pepo cultivars with extremely different fruit size, ‘Munchkin’ (RS) x ‘Big

425 Moose’ (SC) displayed remarkable alterations in fruit size as it resulted in a dramatic decrease in

426 fruit mass and a reduction in both equatorial and polar fruit diameter. This is the first time that

427 intra-species grafting in a Cucurbita species results in changes in fruit morphology. However,

428 grafting has been associated with changes in a number of fruit quality characteristics in the past.

429 Pepper is one of the plants where fruit shape changes attributed to grafting were early recorded

430 and actually found to be inherited to the grafting progenies introducing the whole concept of

431 grafting-induced phenotypic variation) (Taller et al. 1999; Tsaballa et al. 2012; Yagishita &

432 Hirata 1987). (Taller et al. 1999) managed to produce pepper varieties with stable but different

433 fruit characteristics such as fruit shape and flavour from the graft-induced variants of an intra-

434 species grafted plant. In our previous work we have found that grafting-induced fruit shape

435 changes were stably inherited to the grafting progenies when a round-fruited pepper cultivar was

436 grafted on a long-fruited pepper cultivar; scion’s fruits in this case resembled more the fruit

437 shape of the rootstock than that of the scion (Tsaballa et al. 2012). These findings support the

438 already formed idea of graft ‘hybridization’ that involves the movement of genetic material from

439 the rootstock to the scion, a process also called graft transformation and results in changes that

440 are heritable to the scion’s progenies (Goldschmidt 2014; Mudge et al. 2009). It was recently

441 found that whole nuclear genomes can be transferred between plant cells during grafting creating

442 new allopolyploid species (Fuentes et al. 2014). Our results support the notion that grafting is

443 associated with genetic changes in the phenotype of the grafted plants in a way that it could also

444 be used for the introduction of new characteristics in crops.

445 Apart from genetic changes leading to phenotypic changes, agronomical traits such as

446 yield, plant architecture, fruit morphology, fruit quality, as well as the response to abiotic stress

447 or resistance to diseases can be affected substantially by changes in DNA methylation, histone

448 post-translational modifications and miRNAs, all leading to profound changes in gene expression

449 (Dong et al. 2013; Fujimoto et al. 2012; Tsaftaris et al. 2012; Zhong et al. 2013). Likewise, the

450 same traits can be affected by grafting, while increasing evidence has indicated that grafting may

451 induce DNA methylation-pattern alterations. Global DNA methylation patterns were found

452 significantly altered in Solanaceae and Cucurbitaceae inter-species grafting (Avramidou et al.


453 2014; Wu et al. 2013). In Solanaceae species grafting, scion genomic loci with altered DNA

454 methylation patterns contained genes associated to diverse cellular functions while a series of

455 genes encoding DNA methylation-related enzymes were found to have altered gene expression

456 patterns (Wu et al. 2013). In addition, a portion of DNA methylation changes were found to be

457 inherited in the self-pollinated progeny (Wu et al. 2013). Changes in DNA methylation patterns

458 were also shown in grafts between Brassica juncea and B. oleraceae and it was suggested that

459 they are possibly linked to the phenotypic variability observed although in these species it was

460 found that the changes can be both heritable and reversible in the subsequent generations (Cao et

461 al. 2016). In our study, we have noticed significant changes in the DNA methylation pattern of

462 the grafted plants. In the ‘Munchkin’ (RS) x ‘Big Moose’ (SC) combination which resulted in a

463 dramatic fruit size reduction, de-novo DNA methylation levels were markedly increased,

464 suggesting that graft-associated DNA methylation alterations in certain loci may be responsible

465 for altering fruit shape in the scion. On the other hand, in the reciprocal combination, ‘Big

466 Moose’ (RS) x ‘Munchkin’ (SC), a significant increase in DNA demethylation levels was also

467 detected. Although fruit morphology changes were not evidenced in this combination it is

468 possible that increased DNA demethylation has affected other loci associated with fruit quality,

469 and/or environmental responses. Further experiments are needed in order to elucidate the effect

470 of enhanced DNA demethylation in these grafts. Fruit shape changes on grafted plants was also

471 found to be accompanied by alterations in the DNA methylation patterns of scion leaves as

472 shown by the results of ‘Princess’ (RS) x ‘Round green’ (SC) and the reciprocal graft

473 methylation patterns.

474 Changes in DNA methylation of the graft partners could result in profound changes in

475 gene expression in both scion and rootstock tissues and consequently affect a multitude of

476 cellular pathways. The latter can be associated with distinct phenotypes including shoot

477 architecture, total yield, fruit morphology and quality, and responses to environmental stress. Our

478 data showed that intra-species Cucurbita heterografting altered fruit morphology. In the past we

479 have reported similar results when intra-species grafting in pepper using two cultivars of

480 different shape, cv. ‘Long’ (RS) x cv. ‘Round green’ (SC) resulted in a change in scion’s fruit

481 morphology as the fruits had acquired a more elongated shape compared to the control s and the

482 homografted plants, resembling more the fruit phenotype of the rootstock rather than the scion.

483 Taking into account all the above considerations, it is possible that fruit morphology changes in


484 inter-species heterografting of Cucurbitaceae could be associated with DNA methylation

485 alterations in scion tissues that in turn affect gene expression programs linked to fruit

486 morphology traits.

487 However, epigenetic changes in gene expression that may result in phenotypic variation

488 are not only produced by changes in DNA methylation but by other epigenetic mechanisms

489 linked to small RNAs. The 21nt small RNA class of miRNAs has been shown to play crucial

490 roles in plant growth and development controlling seed, vegetative, flowering, reproductive and

491 developmental processes and the responses to abiotic and biotic stress (Li et al. 2016; Zhang

492 2015). In addition, changes in miRNA expression have been demonstrated recently to take place

493 during grafting. MiRNA transcriptomic analyses in Cucurbitaceae grafted plants have revealed

494 that groups of conserved miRNAs such as miR156, miR159, miR164, miR165/166, miR171,

495 miR172, were either induced or repressed in inter-species hetero-grafted scions implying a

496 regulatory role of miRNAs in the grafting process (Li et al. 2013). In our experiments, miR159,

497 miR164, miR166 and miR171 showed significant downregulation in the ‘Munchkin’ (RS) x ‘Big

498 Moose’ (SC) heterograft that resulted in the changed, smaller fruit. These results point out that

499 there is a graft-induced miRNA controlled epigenetic mechanism that possibly underlies fruit

500 development in grafted scions. MiR156 targets plant specific transcription factors of the

501 SQUAMOSA-promoter binding protein (SBP) type, or SBP-like (SPL) type, as well as

502 APETALA2 (AP2) which have been associated with flowering time and organ size (Kim et al.

503 2012; Wang et al. 2009; Wang et al. 2008; Zhang et al. 2011). MiRNA 159 has been implicated

504 in floral meristem development by targeting SPL transcription factors which in turn regulate

505 SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1), LFY and AP1 genes

506 involved in SAM cell differentiation and floral meristem initiation (Achard et al. 2004). In

507 addition, miR159 mediates floral meristem formation by regulating MYB family transcription

508 factors and in particular gibberillin (GA)-specific MYBs. Loss of function

509 of mir159a and mir159b results in the abnormal expression of MYB33 and MYB65 which

510 influences plant development and decreases cell proliferation in leaves (Allen et al. 2010; Allen

511 et al. 2007). Mir164 controls proper formation and separation of adjacent plant organs by

512 targeting genes coding for the NON APICAL MERISTEM (NAM), ATAF and NAC family of

513 transcription factors. These genes regulate the establishment and maintenance of the shoot apical

514 and axillary meristem during floral development and control the formation of floral organs and


515 organ boundaries (Aida & Tasaka 2006; Baker et al. 2005; Kusumanjali et al. 2012; Mallory et

516 al. 2004). MiRNA172 targets the transcription factor gene AP2 and regulates floral meristem

517 initiation, flower patterning and floral organ identity (Chen 2004; Li & Zhang 2015). In addition,

518 miR172 which is regulated by FRUITFULL and AUXIN RESPONSE FACTOR positively

519 controls carpel size via repression of AP2 (Ripoll et al. 2015). MiRNA171 targets the GRAS

520 family transcription factors that play a key role in meristem maintenance, shoot development and

521 flowering time (Wang et al. 2010). Overexpression of miR171 in Arabidopsis led to altered plant

522 height, short branching, root elongation, leaf shape and flower structure (Wang et al. 2010).

523 Overexpression of miR171 in rice and barley had an effect on phase transitions and floral

524 meristem determinacy (Curaba et al. 2013; Fan et al. 2015), whereas overexpression of miR171

525 in tomato resulted in taller plants and earlier flowering (Huang et al. 2013). In conclusion all the

526 miRNAs that were found to be downregulated in the grafted plants we used have been shown to

527 be implicated directly or indirectly in processes linked to cell, meristem, flower and fruit

528 development. Changes in expression of such important miRNA molecules may have huge

529 implications in fruit morphology and may be responsible for the altered phenotypes of the

530 grafted plants. It is possible that some of these miRNA expression changes control key-genes in

531 the fruit growth process that result in grafted plant’s fruit phenotypic change.

532 Apart from the transcriptomic level was studied the grafting effect on the qualitative and

533 quantitative profile of secondary metabolites, mainly polyphenols, of C. pepo fruit samples. A

534 fast, efficient and sensitive state of the art targeted LC-MS/MS analysis was used for this

535 purpose. In the samples used, seventeen phenolic metabolites were identified in comparison to

536 (Iswaldi et al. 2013) who reported twenty-five phenolic compounds obtained from two green

537 zucchini varieties. To a certain extend qualitative differences may arise due to different technical

538 issues: extraction methods, plant material used (genotype, tissue), environmental factors

539 (altitude, climate, rainfall), abiotic stress, drought, UV-radiation, salt stress and nutrient

540 availability, harvesting season, and/or postharvest processing of fruits fruits (Boeing et al. 2014;

541 Cheynier et al. 2013; Nayak et al. 2015; Tiwari & Cummins 2013). Five additional metabolites

542 were identified in this study; elculetin, coniferyl and sinapoyl alcohol, arbutin, carnosic acid and

543 carnosol, even as traces. In fact, to the best of our knowledge this is the first report that the last

544 two diterpenes (carnosic acid and carnosol) are detected in C. pepo, except for Rosmarinus and

545 Salvia species (Birtić et al. 2015; Sarrou et al. 2016). The promotive effect of grafting on


546 biochemical and nutritional traits of some vegetables such as tomato, watermelon and eggplant

547 has been previously reported (Krumbein & Schwarz 2013; Kyriacou et al. 2015; Nicoletto et al. 2013;

548 Soteriou et al. 2014). In the majority of these reports the observed changes were also indirectly

549 connected to the belated harvest point (due to rootstock effect), the seasonal change and the

550 postharvest storage too. According to the data obtained, the cultivar that is used as the scion to

551 the heterografts plays a crucial role for the biosynthesis of the phenolic metabolites. That became

552 apparent, as compounds that were not present in the control s or the homograft of ‘Big Moose’,

553 were detected in the heterograft of ‘Big Moose’ x ‘Munchkin’, while ‘Munchkin’ contained such

554 phenolic compounds in relative concentrations as well. Thus, the changes observed in the

555 phenolic profile of ‘Big Moose’ (RS) x ‘Munchkin’ (SC) and ‘Munchkin’ (RS) x ‘Big Moose’

556 (SC) are more likely scion-dependent than rootstock-dependent. These observations are in

557 agreement with previous studies on Solanaceae species that also reported the main scion effect

558 on final yield and fruit quality traits in grafted plants, while rootstock effects can alter such

559 characteristics possibly due to changes in vigor and concentration of plant growth regulators

560 induced by the rootstocks (Gisbert et al. 2012; Gisbert et al. 2011; Moncada et al. 2013; Muñoz-

561 Falcón et al. 2008; Passam et al. 2005). Furthermore, the ‘trend’ in the accumulation of phenolics

562 seems to be also genetically controlled as in green fruit C. pepo cultivars and more complex than

563 yellow ones.

564 Previous investigations in Cucurbita inter-species heterografting involving significant

565 DNA methylation changes in melon and cucumber scions grafted onto pumpkin rootstocks has

566 been expanded by the current study (Avramidou et al. 2014).

567

568 Conclusion

569 Herein, for the first time, has been demonstrated that intra-species grafting among C. pepo

570 cultivars induced dramatic changes in scion fruit size and that these changes are accompanied by

571 significant alterations in global DNA methylation patterns of scion genomes, miRNA expression

572 changes, and phenolic profile changes. Moreover, it is shown that intra-species C. pepo grafting

573 induced moderate alterations in fruit shape with concomitant substantial changes in scion DNA

574 methylation and miRNA expression patterns. These investigations contribute to a deeper

575 understanding of grafting in Cucurbitaceae and will shed light to the poorly understood

576 molecular mechanisms underlying in the effect of grafting on fruit morphology. Moreover, they


577 will help in understanding the association between graft-induced DNA methylation, miRNA

578 expression and altered phenolic profiles in fruits of grafted plants. Taken together, the

579 differential epigenetic marks and phenolic patterns induced by grafting may lead to the

580 development of epi-molecular markers and metabolic predictors for rapid and efficient selection

581 of grafted vegetables with desired traits of agronomical relevance.

582 References

583 Achard P, Herr A, Baulcombe DC, and Harberd NP. 2004. Modulation of floral development by a

584 gibberellin-regulated microRNA. Development 131:3357-3365.

585 Aida M, and Tasaka M. 2006. Genetic control of shoot organ boundaries. Current opinion in plant

586 biology 9:72-77.

587 Albacete A, Martinez-Anddujar C, Martinez-Perez An, Thompson AJ, Dodd IC, and Perez-Alfocea F.

588 2015. Unravelling rootstock—scion interactions to improve food security. Journal of

589 Experimental Botany. 10.1093/jxb/erv027

590 Allen RS, Li J, Alonso-Peral MM, White RG, Gubler F, and Millar AA. 2010. MicroR159 regulation of

591 most conserved targets in Arabidopsis has negligible phenotypic effects. Silence 1:18.

592 Allen RS, Li J, Stahle MI, Dubroué A, Gubler F, and Millar AA. 2007. Genetic analysis reveals

593 functional redundancy and the major target genes of the Arabidopsis miR159 family. Proceedings

594 of the National Academy of Sciences 104:16371-16376.

595 Avramidou E, Kapazoglou A, Aravanopoulos FA, Xanthopoulou A, Ganopoulos I, Tsaballa A, Madesis

596 P, Doulis AG, and Tsaftaris A. 2014. Global DNA methylation changes in Cucurbitaceae inter-

597 species grafting. Crop Breeding and Applied Biotechnology 15:112-116.

598 Baker CC, Sieber P, Wellmer F, and Meyerowitz EM. 2005. The early extra petals1 mutant uncovers a

599 role for microRNA miR164c in regulating petal number in Arabidopsis. Current Biology 15:303-

600 315.

601 Barrera-Figueroa BE, Wu Z, and Liu R. 2013. Abiotic stress-associated microRNAs in plants: discovery,

602 expression analysis, and evolution. Frontiers in biology 8:189-197.

603 Birtić S, Dussort P, Pierre F-X, Bily AC, and Roller M. 2015. Carnosic acid. Phytochemistry 115:9-19.

604 Boeing JS, Barizão ÉO, e Silva BC, Montanher PF, de Cinque Almeida V, and Visentainer JV. 2014.

605 Evaluation of solvent effect on the extraction of phenolic compounds and antioxidant capacities

606 from the berries: application of principal component analysis. Chemistry Central Journal 8:1-9.

607 Cao L, Yu N, Li J, Qi Z, Wang D, and Chen L. 2016. Heritability and Reversibility of DNA Methylation

608 Induced by in vitro Grafting between Brassica juncea and B. oleracea. Scientific reports 6.


609 Chen X. 2004. A microRNA as a translational repressor of APETALA2 in Arabidopsis flower

610 development. Science 303:2022-2025.

611 Cheynier V, Comte G, Davies KM, Lattanzio V, and Martens S. 2013. Plant phenolics: recent advances

612 on their biosynthesis, genetics, and ecophysiology. Plant Physiology and Biochemistry 72:1-20.

613 Cohen R, Burger Y, Horev C, Koren A, and Edelstein M. 2007. Introducing grafted cucurbits to modern

614 agriculture: the Israeli experience Plant dis 91:916-923.

615 Colla G, Rouphael Y, Leonardi C, and Bie Z. 2010. Role of grafting in vegetable crops grown under

616 saline conditions. Scientia Horticulturae 127:147-155.

617 http://dx.doi.org/10.1016/j.scienta.2010.08.004

618 Curaba J, Talbot M, Li Z, and Helliwell C. 2013. Over-expression of microRNA171 affects phase

619 transitions and floral meristem determinancy in barley. BMC plant biology 13:1.

620 Dong H, Lei J, Ding L, Wen Y, Ju H, and Zhang X. 2013. MicroRNA: function, detection, and

621 bioanalysis. Chemical reviews 113:6207-6233.

622 Fan T, Li X, Yang W, Xia K, Ouyang J, and Zhang M. 2015. Rice osa-miR171c mediates phase change

623 from vegetative to reproductive development and shoot apical meristem maintenance by

624 repressing four OsHAM transcription factors. PloS one 10:e0125833.

625 Fuentes I, Stegemann S, Golczyk H, Karcher D, and Bock R. 2014. Horizontal genome transfer as an

626 asexual path to the formation of new species. Nature 511:232-235.

627 Fujimoto R, Sasaki T, Ishikawa R, Osabe K, Kawanabe T, and Dennis ES. 2012. Molecular Mechanisms

628 of Epigenetic Variation in Plants. International Journal of Molecular Sciences 13:9900-9922.

629 Gisbert C, Prohens J, and Nuez F. 2012. Performance of eggplant grafted onto cultivated, wild, and

630 hybrid materials of eggplant and tomato. International Journal of Plant Production 5:367-380.

631 Gisbert C, Prohens J, Raigón MD, Stommel JR, and Nuez F. 2011. Eggplant relatives as sources of

632 variation for developing new rootstocks: Effects of grafting on eggplant yield and fruit apparent

633 quality and composition. Scientia Horticulturae 128:14-22.

634 Goldschmidt EE. 2014. Plant grafting: new mechanisms, evolutionary implications. Frontiers in plant

635 science 5.

636 Haroldsen V, Szczerba MW, Aktas H, Lopez J, Odias MJ, Chi-Ham CL, Labavitch J, Bennett AB, and

637 Powell ALT. 2012. Mobility of transgenic nucleic acids and proteins within grafted rootstocks for

638 agricultural improvement. Frontiers in plant science 3:39.

639 Huang Y, Bie Z, Liu P, Niu M, Zhen A, Liu Z, Lei B, Gu D, Lu C, and Wang B. 2013. Reciprocal

640 grafting between cucumber and pumpkin demonstrates the roles of the rootstock in the

641 determination of cucumber salt tolerance and sodium accumulation. Scientia Horticulturae

642 149:47-54. http://dx.doi.org/10.1016/j.scienta.2012.04.018


http://dx.doi.org/10.1016/j.scienta.2010.08.004


643 Iswaldi I, Gómez-Caravaca AM, Lozano-Sánchez J, Arráez-Román D, Segura-Carretero A, and

644 Fernández-Gutiérrez A. 2013. Profiling of phenolic and other polar compounds in zucchini

645 (Cucurbita pepo L.) by reverse-phase high-performance liquid chromatography coupled to

646 quadrupole time-of-flight mass spectrometry. Food Research International 50:77-84.

647 Kalantidis K, Schumacher HT, Alexiadis T, and Helm JM. 2008. RNA silencing movement in plants.

648 Biology of the Cell 100:13-26.

649 Katayama S, Ohno F, Yamauchi Y, Kato M, Makabe H, and Nakamura S. 2013. Enzymatic synthesis of

650 novel phenol acid rutinosides using rutinase and their antiviral activity in vitro. Journal of

651 agricultural and food chemistry 61:9617-9622.

652 Kehr J, and Buhtz A. 2008. Long distance transport and movement of RNA through the phloem. Journal

653 of experimental botany 59:85-92.

654 Kim JJ, Lee JH, Kim W, Jung HS, Huijser P, and Ahn JH. 2012. The microRNA156-SQUAMOSA

655 PROMOTER BINDING PROTEIN-LIKE3 module regulates ambient temperature-responsive

656 flowering via FLOWERING LOCUS T in Arabidopsis. Plant Physiology 159:461-478.

657 King SR, Davis AR, Liu W, and Levi A. 2008. Grafting for Disease Resistance. HortScience 43:1673-

658 1676.

659 Krumbein A, and Schwarz D. 2013. Grafting: A possibility to enhance health-promoting and flavour

660 compounds in tomato fruits of shaded plants? Scientia Horticulturae 149:97-107.

661 Kubota C, McClure MA, Kokalis-Burelle N, Bausher MG, and Rosskopf EN. 2008. Vegetable Grafting:

662 History, Use, and Current Technology Status in North America. HortScience 43:1664-1669.

663 Kusumanjali K, Kumari G, Srivastava PS, and Das S. 2012. Sequence conservation and divergence in

664 miR164C1 and its target, CUC1, in Brassica species. Plant biotechnology reports 6:149-163.

665 Kyriacou MC, Soteriou GA, Rouphael Y, Siomos AS, and Gerasopoulos D. 2015. Configuration of

666 watermelon fruit quality in response to rootstock‐mediated harvest maturity and postharvest

667 storage. Journal of the Science of Food and Agriculture.

668 Lee J-M, Kubota C, Tsao SJ, Bie Z, Echevarria PH, Morra L, and Oda M. 2010. Current status of

669 vegetable grafting: Diffusion, grafting techniques, automation. Scientia Horticulturae 127:93-

670 105. http://dx.doi.org/10.1016/j.scienta.2010.08.003

671 Li C, and Zhang B. 2015. MicroRNAs in Control of Plant Development. Journal of Cellular Physiology

672 231:303-313. 10.1002/jcp.25125

673 Li S, Castillo‐Gonzalez C, Yu B, and Zhang X. 2016. The functions of plant small RNAs in development

674 and in stress responses. The Plant Journal.



675 Li X, Bian H, Song D, Ma S, Han N, Wang J, and Zhu M. 2013. Flowering time control in ornamental

676 gloxinia (Sinningia speciosa) by manipulation of miR159 expression. Annals of botany 111:791-

677 799.

678 Lough TJ, and Lucas WJ. 2006. Integrative plant biology: role of phloem long-distance macromolecular

679 trafficking. Annu Rev Plant Biol 57:203-232.

680 Louws FJ, Rivard CL, and Kubota C. 2010. Grafting fruiting vegetables to manage soilborne pathogens,

681 foliar pathogens, arthropods and weeds. Scientia horticulturae 127:127-146.

682 Lucas WJ, Yoo B-C, and Kragler F. 2001. RNA as a long-distance information macromolecule in plants.

683 Nature Reviews Molecular Cell Biology 2:849-857.

684 Mallory AC, Dugas DV, Bartel DP, and Bartel B. 2004. MicroRNA regulation of NAC-domain targets is

685 required for proper formation and separation of adjacent embryonic, vegetative, and floral organs.

686 Current Biology 14:1035-1046.

687 Melnyk CW, Molnar A, and Baulcombe DC. 2011. Intercellular and systemic movement of RNA

688 silencing signals. The EMBO journal 30:3553-3563.

689 Mermigka G, Verret F, and Kalantidis K. 2015. RNA silencing movement in plants. Journal of

690 integrative plant biology.

691 Mohamed F, El-Hamed K, Elwan M, and Hussien M. 2012. Impact of Grafting on Watermelon Growth,

692 Fruit Yield and Quality. Vegetable Crops Research Bulletin 76.

693 Moncada A, Miceli A, Vetrano F, Mineo V, Planeta D, and D’Anna F. 2013. Effect of grafting on yield

694 and quality of eggplant (Solanum melongena L.). Scientia Horticulturae 149:108-114.

695 Mudge K, Janick J, Scofield S, and Goldschmidt EE. 2009. A History of Grafting. Horticultural reviews:

696 John Wiley & Sons, Inc., 437-493.

697 Muñoz-Falcón JE, Prohens J, Rodríguez-Burruezo A, and Nuez F. 2008. Potential of local varieties and

698 their hybrids for the improvement of eggplant production in the open field and greenhouse

699 cultivation. Journal of Food Agriculture and Environment 6:83.

700 Nascimento PLA, Nascimento TCES, Ramos NSM, Silva GR, Gomes JEG, Falcão REA, Moreira KA,

701 Porto ALF, and Silva T. 2014. Quantification, antioxidant and antimicrobial activity of phenolics

702 isolated from different extracts of Capsicum frutescens (Pimenta Malagueta). Molecules 19:5434-

703 5447.

704 Nayak B, Liu RH, and Tang J. 2015. Effect of processing on phenolic antioxidants of fruits, vegetables,

705 and grains—A review. Critical reviews in food science and nutrition 55:887-918.

706 Nicoletto C, Tosini F, and Sambo P. 2013. Effect of grafting on biochemical and nutritional traits of

707 ‘Cuore di Bue’tomatoes harvested at different ripening stages. Acta Agriculturae Scandinavica,

708 Section B–Soil & Plant Science 63:114-122.


709 Obrero An, Die JV, Román Bn, Gómez P, Nadal S, and González-Verdejo CI. 2011. Selection of

710 reference genes for gene expression studies in zucchini (Cucurbita pepo) using qPCR. Journal of

711 agricultural and food chemistry 59:5402-5411.

712 Omid A, Keilin T, Glass A, Leshkowitz D, and Wolf S. 2007. Characterization of phloem-sap

713 transcription profile in melon plants. Journal of experimental botany 58:3645-3656.

714 Orsini F, Sanoubar R, Oztekin GB, Kappel N, Tepecik M, Quacquarelli C, Tuzel Y, Bona S, and

715 Gianquinto G. 2013. Improved stomatal regulation and ion partitioning boosts salt tolerance in

716 grafted melon. Functional Plant Biology 40:628-636. http://dx.doi.org/10.1071/FP12350

717 Palauqui JC, Elmayan T, Pollien JM, and Vaucheret H. 1997. Systemic acquired silencing:

718 transgene‐specific post‐transcriptional silencing is transmitted by grafting from silenced stocks to

719 non‐silenced scions. The EMBO Journal 16:4738-4745.

720 Passam HC, Stylianou M, and Kotsiras A. 2005. Performance of eggplant grafted on tomato and eggplant

721 rootstocks. European Journal of Horticultural Science:130-134.

722 Pfaffl MW, Horgan GW, and Dempfle L. 2002. Relative expression software tool (REST©) for group-

723 wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic

724 acids research 30:e36-e36.

725 Ripoll JJ, Bailey LJ, Mai Q-A, Wu SL, Hon CT, Chapman EJ, Ditta GS, Estelle M, and Yanofsky MF.

726 2015. microRNA regulation of fruit growth. Nature plants 1.

727 Rouphael Y, Cardarelli M, Bassal A, Leonardi C, Giuffrida F, and Colla G. 2012. Vegetable quality as

728 affected by genetic, agronomic and environmental factors. Journal of Food, Agriculture &

729 Environment 10:680-688.

730 Sarrou E, Martens S, and Chatzopoulou P. 2016. Metabolite profiling and antioxidative activity of Sage

731 (Salvia fruticosa Mill.) under the influence of genotype and harvesting period. Industrial Crops

732 and Products 94:240-250.

733 Schwab R, Palatnik JF, Riester M, Schommer C, Schmid M, and Weigel D. 2005. Specific effects of

734 microRNAs on the plant transcriptome. Developmental cell 8:517-527.

735 Soteriou GA, Kyriacou MC, Siomos AS, and Gerasopoulos D. 2014. Evolution of watermelon fruit

736 physicochemical and phytochemical composition during ripening as affected by grafting. Food

737 chemistry 165:282-289.

738 Spiegelman Z, Golan G, and Wolf S. 2013. Don’t kill the messenger: long-distance trafficking of mRNA

739 molecules. Plant science 213:1-8.

740 Taller J, Yagishita N, and Hirata Y. 1999. Graft-induced variants as a source of novel characteristics in

741 the breeding of pepper (Capsicum annuum L.). Euphytica 108:73-78.


http://dx.doi.org/10.1071/FP12350

742 Tiwari U, and Cummins E. 2013. Factors influencing levels of phytochemicals in selected fruit and

743 vegetables during pre-and post-harvest food processing operations. Food Research International

744 50:497-506.

745 Tsaballa A, Athanasiadis C, Pasentsis K, Ganopoulos I, Nianiou-Obeidat I, and Tsaftaris A. 2012.

746 Molecular studies of inheritable grafting induced changes in pepper (Capsicum annuum) fruit

747 shape. Scientia Horticulturae 149:2-8. http://dx.doi.org/10.1016/j.scienta.2012.06.018

748 Tsaftaris AS, Kapazoglou A, and Darzentas N. 2012. Epigenetics, Epigenomics, and Implications in Plant

749 Breeding "Plant biotechnology and agriculture: Prospects for the 21st century" 2012. eds A.

750 Altman and P.M. Haegawa Elsevier Press.

751 Valenzuela GM, Soro AS, Tauguinas AL, Gruszycki MR, Cravzov AL, Giménez MC, and Wirth A.

752 2014. Evaluation polyphenol content and antioxidant activity in extracts of Cucurbita spp. Open

753 Access Library Journal 1:1.

754 Varkonyi-Gasic E, Wu R, Wood M, Walton EF, and Hellens RP. 2007. Protocol: a highly sensitive RT-

755 PCR method for detection and quantification of microRNAs. Plant methods 3:12.

756 Vekemans X, Beauwens T, Lemaire M, and Roldán‐Ruiz I. 2002. Data from amplified fragment length

757 polymorphism (AFLP) markers show indication of size homoplasy and of a relationship between

758 degree of homoplasy and fragment size. Molecular ecology 11:139-151.

759 Vos P, Hogers R, Bleeker M, Reijans M, Van de Lee T, Hornes M, Friters A, Pot J, Paleman J, and

760 Kuiper M. 1995. AFLP: a new technique for DNA fingerprinting. Nucleic acids research

761 23:4407-4414.

762 Vrhovsek U, Masuero D, Gasperotti M, Franceschi P, Caputi L, Viola R, and Mattivi F. 2012. A versatile

763 targeted metabolomics method for the rapid quantification of multiple classes of phenolics in

764 fruits and beverages. Journal of agricultural and food chemistry 60:8831-8840.

765 Wang J-W, Czech B, and Weigel D. 2009. miR156-regulated SPL transcription factors define an

766 endogenous flowering pathway in Arabidopsis thaliana. cell 138:738-749.

767 Wang J-W, Schwab R, Czech B, Mica E, and Weigel D. 2008. Dual effects of miR156-targeted SPL

768 genes and CYP78A5/KLUH on plastochron length and organ size in Arabidopsis thaliana. The

769 plant cell 20:1231-1243.

770 Wang L, Mai Y-X, Zhang Y-C, Luo Q, and Yang H-Q. 2010. MicroRNA171c-targeted SCL6-II, SCL6-

771 III, and SCL6-IV genes regulate shoot branching in Arabidopsis. Molecular plant 3:794-806.

772 Wu R, Wang X, Lin Y, Ma Y, Liu G, Yu X, Zhong S, and Liu B. 2013. Inter-Species Grafting Caused

773 Extensive and Heritable Alterations of DNA Methylation in <italic>Solanaceae</italic> Plants.

774 PloS one 8:e61995.



775 Xanthopoulou A, Ganopoulos I, Kalivas A, Nianiou-Obeidat I, Ralli P, Moysiadis T, Tsaftaris A, and

776 Madesis P. 2015. Comparative analysis of genetic diversity in Greek Genebank collection of

777 summer squash (Cucurbita pepo) landraces using start codon targeted (SCoT) polymorphism and

778 ISSR markers. Australian Journal of Crop Science 9:14.

779 Yagishita N, and Hirata Y. 1987. Graft-induced change in fruit shape in Capsicum annuum LI genetic

780 analysis by crossing. Euphytica 36:809-814.

781 Yetisir H, Özdemir E, Aras V, Candır E, and Aslan H. 2013. Rootstocks effect on plant nutrition

782 concentration in different organ of grafted watermelon Agricultural Sciences 4 230-237

783 Zhang B. 2015. MicroRNA: a new target for improving plant tolerance to abiotic stress. Journal of

784 experimental botany 66:1749-1761.

785 Zhang X, Zou Z, Zhang J, Zhang Y, Han Q, Hu T, Xu X, Liu H, Li H, and Ye Z. 2011. Over-expression

786 of sly-miR156a in tomato results in multiple vegetative and reproductive trait alterations and

787 partial phenocopy of the sft mutant. FEBS letters 585:435-439.

788 Zhong S, Fei Z, Chen Y-R, Zheng Y, Huang M, Vrebalov J, McQuinn R, Gapper N, Liu B, Xiang J, Shao

789 Y, and Giovannoni JJ. 2013. Single-base resolution methylomes of tomato fruit development

790 reveal epigenome modifications associated with ripening. Nat Biotech 31:154-159.

791

792


793 Figure Legends

794 Figure 1: Fruits of cv. ‘Munchkin’ and cv. ‘Big Moose’ grafted plants and their controls

795 (A) Control non-grafted ‘Munchkin’ (left) and ‘Big Moose’ (right)

796 (B) Grafted ‘Big Moose’ (SC) on ‘Munchkin’ (RS) (top) and reciprocal combination (bottom)

797 with no change in fruit morphology that is the same as the fruit morphology of the non-grafted

798 scions.

799 (C) Grafted ‘Big Moose’ (SC) on cv. ‘Munchkin’ (RS) with the altered fruit morphology

800 growing on the grafted plant.

801 (D) Grafted ‘Big Moose’ (SC) on cv. ‘Munchkin’ (RS) with the altered fruit morphology (left)

802 next to grafted ‘Big Moose’ (SC) on cv. ‘Munchkin’ (RS) with no alteration (right).

803

804 Figure 2: Fruits, flowers and leaves of grafted plants cv. ‘Round green’, cv. ‘Princess’ and

805 their controls.

806 (A) Control non-grafted ‘Round green’

807 (B) Grafted ‘Round green’ (SC) on ‘Princess’ (RS)

808 (C) Control non-grafted ‘Princess’

809 (D) Grafted ‘Princess’ (SC) on ‘Round green’ (RS)

810

811 Figure 3: Relative (by comparison to non-grafted seedlings) DNA methylation and

812 demethylation levels (percent of total within each sample; averaged) of grafted plants based

813 on MSAP analysis. Relative levels at randomly sampled 5’- CCGG -3’sites with the MSAP

814 marker (A) ‘Munchkin’ x ‘Big Moose’ grafted plants versus ‘Big Moose’ s. B) ‘Big Moose’ x

815 ‘Munchkin’ plants versus ‘Munchkin’ s. C) ‘Munchkin’ x ‘Big Moose’ grafted plant with fruit

816 size changes versus ‘Big Moose’ seedlings. Asterisks indicate values significantly lower than the

817 values of the corresponding seedling controls (P<0.05).y-axis: relative (percent) methylation

818 level.

819

820 Figure 4: Relative (by comparison to non-grafted seedlings) levels at randomly sampled 5’-

821 CCGG -3’sites with MSAP markers (A) ‘Round green’ x ‘Princess grafted plants versus

822 ‘Princess’ seedlings. B) ‘Princess’ x ‘Round green’ plants versus ‘Round green’ seedlings

823 (percent of total within each sample; averaged). Asterisks indicate values significantly lower


824 than the values of the corresponding seedling controls (P<0.05).y-axis: relative (percent)

825 methylation level.

826 Figure 5: Quantitative qRT-PCR analyses of selected miRNAs in grafted plants and their

827 non-grafted controls

828 (A) miRNA expression in heterografts of ‘Big moose’ and ‘Munchkin’. B= ‘Big Mouse’, M=

829 ‘Munchkin’.

830 (B) miRNA expression levels in heterografts of ‘Princess’ and ‘Round green’. P= ‘Princess’,

831 R= ‘Round green’

832 Expression values were normalized to those of the reference gene CpEF-1a. Relative expression

833 ratio of each sample was compared to the control group which was assigned arbitrarily the value

834 of 1. Data represent mean values from two independent experiments with standard deviations.

835 Values significantly different (P < 0.05) from the control sample (‘Big Moose’) are marked with

836 an asterisk (*).

837

838 Figure 6: Overlay heat map of metabolite profiles in grafted plants in comparison with

839 non-grafted controls.

840 (A) For the cultivars that differ in fruit size and their grafted plants. On the left the part of the

841 heatmap that corresponds to the graftings and the control that produced small fruits in terms of

842 size (red color), on the right the part of the heatmap that corresponds to the ones that produced

843 large fruit (pink color).

844 (B) For the cultivars that differ in fruit shape and their grafted plants. On the left the part of the

845 heatmap that corresponds to the graftings and the control that produced round fruits (red color),

846 on the right the part of the heatmap that corresponds to the ones that produced elongated fruits

847 (pink color). The metabolites in each square represent the effect of plant grafting on the amount

848 of every metabolite using a false-color scale.

849


Figure 1(on next page)

Fruits of cv. ‘Munchkin’ and cv. ‘Big Moose’ grafted plants and their controls.

Figure 1: Fruits of cv. ‘Munchkin’ and cv. ‘Big Moose’ grafted plants and their controls (A)

Control non-grafted ‘Munchkin’ (left) and ‘Big Moose’ (right) (B) Grafted ‘Big Moose’ (SC) on

‘Munchkin’ (RS) (top) and reciprocal combination (bottom) with no change in fruit

morphology that is the same as the fruit morphology of the non-grafted scions. (C) Grafted

‘Big Moose’ (SC) on cv. ‘Munchkin’ (RS) with the altered fruit morphology growing on the

grafted plant. (D) Grafted ‘Big Moose’ (SC) on cv. ‘Munchkin’ (RS) with the altered fruit

morphology (left) next to grafted ‘Big Moose’ (SC) on cv. ‘Munchkin’ (RS) with no alteration

(right).


Munchkin BigMoose

MunchkinxBigMoose

fruitchangedplant MunchkinxBigMoose

A)

C) D)

MunchkinxBigMoose

BigMoosexMunchkin

B)



: Fruits, flowers and leaves of grafted plants cv. ‘Round green’, cv. ‘Princess’ and their

controls

Figure 2: Fruits, flowers and leaves of grafted plants cv. ‘Round green’, cv.

‘Princess’ and their controls. (A) Control non-grafted ‘Round green’ (B) Grafted ‘Round

green’ (SC) on ‘Princess’ (RS) (C) Control non-grafted ‘Princess’ (D) Grafted ‘Princess’ (SC) on

‘Round green’ (RS)


RoundgreenxPrincessPrincess

Roundgreen PrincessxRoundgreen

A) B)

C) D)



Relative (by comparison to non-grafted seedlings) DNA methylation and demethylation

levels (percent of total within each sample; averaged) of grafted plants based on MSAP

analysis.

Figure 3: Relative (by comparison to non-grafted seedlings) DNA methylation and

demethylation levels (percent of total within each sample; averaged) of grafted

plants based on MSAP analysis. Relative levels at randomly sampled 5’- CCGG -3’sites

with the MSAP marker (A) ‘Munchkin’ x ‘Big Moose’ grafted plants versus ‘Big Moose’ s. B)

‘Big Moose’ x ‘Munchkin’ plants versus ‘Munchkin’ s. C) ‘Munchkin’ x ‘Big Moose’ grafted

plant with fruit size changes versus ‘Big Moose’ seedlings. Asterisks indicate values

significantly lower than the values of the corresponding seedling controls (P<0.05).y-axis:

relative (percent) methylation level.


0

5

10

15

20

25

30

35

40

Nochange Demethyla5on De-novomethyla5on

MunchkinxBigMoosegra@edplantsversusBigMooseseed-plants

0

5

10

15

20

25

30

35


BigMoosexMunchkingra?edplantsversusMunchkinseed-plants

0

5

10

15

20

25

30

35


MunchkinxBigMoosegra?edplantwithfruitsizechangesversusBig

Mooseseed-plant

A)

B)

C)



Relative (by comparison to non-grafted seedlings) levels at randomly sampled 5’- CCGG

-3’sites with MSAP markers

Figure 4: Relative (by comparison to non-grafted seedlings) levels at randomly

sampled 5’- CCGG -3’sites with MSAP markers (A) ‘Round green’ x ‘Princess grafted plants

versus ‘Princess’ seedlings. B) ‘Princess’ x ‘Round green’ plants versus ‘Round green’

seedlings (percent of total within each sample; averaged). Asterisks indicate values

significantly lower than the values of the corresponding seedling controls (P<0.05).y-axis:

relative (percent) methylation level.


0

2

4

6

8

10

12

14

16

18


RoundgreenxPrincessgra@edplantsversusPrincessseed-plants

0

5

10

15

20

25

30


PrincessxRoundgreengra?edplantsversusRoundgreenseed-plants

A)

B)



Quantitative qRT-PCR analyses of selected miRNAs in grafted plants and their non-

grafted controls

Figure 5: Quantitative qRT-PCR analyses of selected miRNAs in grafted plants and

their non-grafted controls (A) miRNA expression in heterografts of ‘Big moose’ and

‘Munchkin’. B= ‘Big Mouse’, M= ‘Munchkin’. (B) miRNA expression levels in heterografts of

‘Princess’ and ‘Round green’. P= ‘Princess’, R= ‘Round green’ Expression values were

normalized to those of the reference gene CpEF-1a. Relative expression ratio of each sample

was compared to the control group which was assigned arbitrarily the value of 1. Data

represent mean values from two independent experiments with standard deviations. Values

significantly different (P < 0.05) from the control sample (‘Big Moose’) are marked with an

asterisk (*).


0

0,2

0,4

0,6

0,8

1

1,2

B MXB M BXM

Rela%

vreexpressionra%

o

miR156

0

0,2

0,4

0,6

0,8

1

1,2

B MXB M BXM

miR159

0

0,2

0,4

0,6

0,8

1

1,2

1,4

B MXB M BXM

miR164

0

0,2

0,4

0,6

0,8

1

1,2

B MXB M BXM

miR166

0

1

2

3

4

B MXB M BXM

miR171

A)

B)



Overlay heat map of metabolite profiles in grafted plants in comparison with non-

grafted controls.

Figure 6: Overlay heat map of metabolite profiles in grafted plants in comparison

with non-grafted controls. (A) For the cultivars that differ in fruit size and their grafted

plants. On the left the part of the heatmap that corresponds to the graftings and the control

that produced small fruits in terms of size (red color), on the right the part of the heatmap

that corresponds to the ones that produced large fruit (pink color). (B) For the cultivars that

differ in fruit shape and their grafted plants. On the left the part of the heatmap that

corresponds to the graftings and the control that produced round fruits (red color), on the

right the part of the heatmap that corresponds to the ones that produced elongated fruits

(pink color). The metabolites in each square represent the effect of plant grafting on the

amount of every metabolite using a false-color scale.


A)

B)


Table 1(on next page)

Data for the three morphological traits in control (non-grafted), homo-grafted and

hetero-grafted pumpkin plants.

Table 1. Data for the three morphological traits in control (non-grafted), homo-grafted and

hetero-grafted pumpkin plants.


1 Table 1. Data for the three morphological traits in control (non-grafted), homo-grafted and

2 hetero-grafted pumpkin plants.

Grafts

(rootstock,

scion),

seedlings (cv.)

‘Big

Moose’‘Munchkin’

‘Big Moose’

x ‘Big

Moose’

‘Munchkin’ x

‘Big Moose’

‘Munchkin’ x

‘Munchkin’

‘Big Moose’

x

‘Munchkin’

‘Munchkin’ x

‘Big Moose’

with altered

fruit size

Weight (kg) 9.71±1.39a 0.18±0.04b 11.73±1.92a 8.78±0.83a 0.12±0.02b 0.15±0.03b 3.7ab

Equatorial

diameter (cm)

37.83±2.36

a8.78±0.92b 40.22±3.62a 36.05±2.09a 8.28±0.67b 8.02±1.03b 19.15b

Polar diameter

(cm)

31.89±5.32

a4.09±0.43b 31.94±8.66a 28.85±5.05a 4.12±0.56b 4.47±0.65b 17.26b

Grafts

(rootstock,

scion),

seedlings (cv.)

‘Princess’‘Round

green’

‘Princess’ x

‘Princess’

‘Princess’ x

‘Round

green’

‘Round

green’ x

‘Round

green’

‘Round

green’ x

‘Princess’

Weight (kg) 0.20±0.04a 0.17±0.03a 0.23±0.05a 0.16±0.13a 0.15±0.14a 0.21±0.06a

Equatorial

diameter (cm)

17.83±1.32

a9.22±0.06b 17.4±1.55a 9.34±0.02b 9.18±0.04b 17.21±1.85a

Polar diameter

(cm)5.81±0.06a 10.34±0.3b 5.93±0.24a 10.12±0.32b 9.92±0.12b 6.42±0.12a

3

4 Different letters in the columns indicate significant differences according to Duncans’ test for P< 0.05

Grafts

(rootstock,

scion),

seedlings (cv.)

‘Big Moose’ ‘Munchkin’

‘Big Moose’

x ‘Big

Moose’

‘Munchkin’ x

‘Big Moose’

‘Munchkin’ x

‘Munchkin’

‘Big Moose’

x

‘Munchkin’

‘Munchkin’ x

‘Big Moose’

with altered

fruit size

Weight (kg) 9.71±1.39a 0.18±0.04b 11.73±1.92a 8.78±0.83ab 0.12±0.02b 0.15±0.03b 3.7ab

Equatorial

diameter (cm)37.83±2.36a 8.78±0.92b 40.22±3.62a 36.05±2.09a 8.28±0.67b 8.02±1.03b 19.15b

Polar diameter

(cm)31.89±5.32a 4.09±0.43b 31.94±8.66a 28.85±5.05ab 4.12±0.56b 4.47±0.65b 17.26b

Grafts

(rootstock,

scion),

seedlings (cv.)

‘Princess’‘Round

green’

‘Princess’ x

‘Princess’

‘Princess’ x

‘Round

green’

‘Round

green’ x

‘Round

green’

‘Round

green’ x

‘Princess’

Weight (kg) 0.20±0.04a 0.17±0.03a 0.23±0.05a 0.16±0.13a 0.15±0.14a 0.21±0.06a

Equatorial

diameter (cm)17.83±1.32a 9.22±0.06b 17.4±1.55a 9.34±0.02b 9.18±0.04b

17.21±1.85a

b

Polar diameter

(cm)5.81±0.06a 10.34±0.3b 5.93±0.24a 10.12±0.32b 9.92±0.12b 6.42±0.12ab

5

6 Different letters in the columns indicate significant differences according to Duncans’ test for P< 0.05

7



The effect of grafting on secondary metabolites of pumpkin related with fruit size.

Table 2: The effect of grafting on secondary metabolites of pumpkin related with fruit size.


1 Table 2: The effect of grafting on secondary metabolites of pumpkin related with fruit size.

Concentration (mg/100 g DW)

Compound Big MooseBig Moose x

Big Moose

Big Moose

x

Munchkin

MunchkinMunchkin x

Munchkin

Munchkin x

Big Moose

1 p-hydroxybenzoic acid 0.000 0.000 0.575 a 0.666 a 0.492 a 0.000

2 ferulic acid 0.042 b 0.004 b 0.264 a 0.083 b 0.060 b 0.000

3 vanillic acid 0.000 0.000 0.039 a 0.000 0.005 b 0.000

4 chlorogenic acid 0.000 0.134 a 0.000 0.105 b 0.000 0.000

5 esculetin 0.016 a 0.010 ab 0.007 ab 0.000 0.006 ab 0.011 ab

6 coniferyl alcohol 0.134 a 0.000 0.137 a 0.140 a 0.056 bc 0.089 ab

7 sinapyl alcohol 0.000 0.000 0.092 b 0.215 a 0.000 0.000

8 naringenin 0.000 0.000 0.027 0.000 0.000 0.000

9 dihydrokaempferol 0.000 0.000 0.000 0.090 0.000 0.000

10 kaempferol-3-O-rutinoside 1.915 b 3.069 a 0.000 0.000 0.000 1.726 b

11quercetin-3-O-glucoside +

quercetin-3-O-galactoside0.077 b 0.053 c 0.000 0.000 0.000 0.405 a

12 rutin 1.598 b 0.678 c 0.000 0.023 d 0.000 2.434 a

13 isorhamnetin 0.000 0.000 0.000 0.000 0.000 0.019

14 isorhamnetin-3-O-glucoside 0.000 0.187 b 0.000 0.000 0.000 0.326 a

15 isorhamnetin-3-O-rutinoside 18.855 a 15.703 b 0.000 0.000 0.000 14.430 b

16 arbutin 0.000 0.000 0.262 a 0.261 a 0.141 b 0.000

17 carnosol 0.104 b 0.165 a 0.081 bc 0.032 d 0.040 d 0.056 cd

18 carnosic acid 0.396 a 0.431 a 0.135 b 0.104 b 0.030 c 0.134 b

Total identified 23.137 20.434 1.618 1.719 0.831 19.630

2 Different letters in the columns indicate significant differences according to Duncans’ multiple range test performed across all grafting treatments (

3 P< 0.05).

4

5



The effect of grafting on secondary metabolites of pumpkin related with fruit shape.

Table 3: The effect of grafting on secondary metabolites of pumpkin related with fruit shape.


1 Table 3: The effect of grafting on secondary metabolites of pumpkin related with fruit shape.

Concentration (mg/100 g DW)

Compound Princess Princess x Princess

Princess x Round

green Round green

Round green x

Round green

Round green x

Princess

1 p-hydroxybenzoic acid 0.671 c 1.073 ab 1.298 a 0.601 c 0.818 bc 1.029 ab

2 cinnamic acid 2.422 a 1.366 b 0.353 c 1.680 b 0.000 1.372 a

3 ferulic acid 2.720 a 0.000 1.888 b 1.494 c 0.086 d 2.805 a

4 vanillic acid 0.818 b 0.000 0.000 0.425 c 0.783 b 1.215 a

5 esculetin 0.006 bc 0.000 0.032 a 0.015 b 0.012 bc 0.006 bc

6 coniferyl alcohol 0.042 b 0.000 0.098 a 0.127 a 0.000 0.000

7 orientin 0.000 0.000 0.038 0.030 0.000 0.000

8 naringenin 0.000 0.000 0.015 0.017 0.000 0.026

9 dihydrokaempferol 0.000 0.017 b 0.000 0.034 a 0.000 0.000

10 kaempferol-3-O-rutinoside 0.000 0.029 b 1.004 a 0.194 b 0.071 b 0.000

11 quercetin 0.000 0.000 0.321 0.000 0.000 0.000

12

quercetin-3-O-glucoside +

quercetin-3-O-galactoside 0.000 0.000 0.416 0.000 0.000 0.000

13 rutin 0.278 c 0.000 21.268 a 2.000 b 0.234 c 0.291 c

14 isorhamnetin 0.000 0.000 0.036 0.000 0.000 0.000

15 isorhamnetin-3-O-glucoside 0.000 0.000 0.202 a 0.066 b 0.000 0.000

16 isorhamnetin-3-O-rutinoside 0.478 cd 0.960 c 16.305 a 9.948 b 0.942 c 0.310 d

17 carnosol 0.052 b 0.002 d 0.151 a 0.039 bc 0.021 bcd 0.006 cd

Total identified 7.487 3.448 43.424 16.671 2.967 7.060

2

3 Different letters in the columns indicate significant differences according to Duncans’ multiple range test performed across all grafting treatments

4 ( P< 0.05).


Global epigenetic and metabolic changes accompany the ...27 Abstract 28 To further understand the...

Documents

Transcript of Global epigenetic and metabolic changes accompany the ...27 Abstract 28 To further understand the...